Analysis of chemically crosslinked cellular samples

Abstract

A method of analyzing cellular samples that include a chemically crosslinked analyte is provided. The analysis typically involves the use of mass spectrometry.

1. A method of analyzing analytes, the method comprising:
 providing a cellular sample comprising chemically crosslinked analytes, wherein the sample is one or more formalin-fixed, paraffin-embedded tissue sections, and wherein the analytes are proteins or peptides or combinations thereof; 
 separating the chemically crosslinked analytes from the formalin-fixed, paraffin-embedded tissue sections by heat; 
 reversing at least a portion of the chemical crosslinks in the crosslinked analytes and forming decrosslinked analytes, wherein said reversing at least a portion of the chemical crosslinks comprises cleaving at least a portion of the chemical crosslinks, with the proviso that substantially no naturally occurring bonds in the analytes 
 are cleaved during said reversing at least a portion of the chemical crosslinks; and 
 analyzing the analytes by analyzing the decrosslinked analytes using mass spectrometry. 
 
     
     
       2. The method of  claim 1  wherein the sample further comprises analytes that are not chemically crosslinked and analyzing both the decrosslinked analytes and the analytes that are not chemically crosslinked. 
     
     
       3. The method of  claim 2  wherein the analytes that are not chemically crosslinked comprise pharmaceuticals, metabolites, or vitamins. 
     
     
       4. The method of  claim 1 , wherein separating the chemically crosslinked analytes from the formalin-fixed, paraffin-embedded tissue occurs prior to said reversing at least a portion of the chemical crosslinks. 
     
     
       5. The method of  claim 1  wherein the crosslinked analytes are crosslinked proteins. 
     
     
       6. The method of  claim 1 , wherein said cleaving at least a portion of the chemical crosslinks is done through the application of an energy in the presence of water or buffer at a range of pH values. 
     
     
       7. The method of  claim 6  wherein the energy is heat. 
     
     
       8. The method of  claim 6  wherein the energy is radiation. 
     
     
       9. The method of  claim 1  wherein said analyzing the decrosslinked analytes comprises cleaving at least a portion of the bonds in the decrosslinked analyte, forming analyte fragments and analyzing the analyte fragments using mass spectrometry. 
     
     
       10. The method of  claim 9  wherein the cleaving at least a portion of the bonds in the decrosslinked analytes comprises contacting the decrosslinked analytes with an enzyme or chemical reagent. 
     
     
       11. The method of  claim 10  wherein the cleaving at least a portion of the bonds in the decrosslinked analytes comprises contacting the decrosslinked analytes with an enzyme. 
     
     
       12. The method of  claim 11  wherein the enzyme is selected from the group consisting of trypsin, pepsin, pronase, and chymotrypsin. 
     
     
       13. The method of  claim 12  wherein the decrosslinked analytes are proteins, the enzyme is trypsin, and the analyte fragments are an eluate comprising protein fragments. 
     
     
       14. The method of  claim 1  wherein the cellular sample is from a plant or animal. 
     
     
       15. The method of  claim 14  wherein the cellular sample is from a human. 
     
     
       16. The method of  claim 14  wherein the cellular sample is from an individual having a disease. 
     
     
       17. The method of  claim 16  wherein the disease is a progressive disease, and the formalin-fixed, paraffin-embedded tissue sections are a plurality of tissue sections representing different stages of the progressive disease. 
     
     
       18. The method of  claim 14  wherein the cellular sample is from a non-human animal that is model for a disease. 
     
     
       19. The method of  claim 14  wherein the cellular sample comprises at least one cell having an exogenous nucleic acid. 
     
     
       20. The method of  claim 1  wherein said analyzing the decrosslinked analytes comprises identifying the decrosslinked analytes. 
     
     
       21. The method of  claim 20  wherein said identifying the decrosslinked analytes comprises:
 reading unique identifiers of the decrosslinked analytes; and 
 determining the identities of the decrosslinked analytes. 
 
     
     
       22. The method of  claim 1  wherein said analyzing the decrosslinked analytes comprise quantifying the decrosslinked analytes. 
     
     
       23. A method of analyzing analytes, the method comprising:
 providing a formalin-fixed, paraffin embedded, tissue section comprising one or more analytes comprising chemically fixed bonds; 
 removing the one or more analytes from the paraffin of the formalin-fixed, paraffin embedded, tissue section; 
 cleaving at least a portion of the chemically fixed bonds in the one or more analytes and forming cleaved analytes, with the proviso that substantially no naturally occurring bonds in the analytes are cleaved; and 
 analyzing the analytes by analyzing the cleaved analytes using mass spectrometry, wherein the one or more analytes are proteins or peptides or combinations thereof. 
 
     
     
       24. The method of  claim 23  further comprising placing the formalin—fixed, paraffin embedded, tissue section on a substrate comprising a polymeric material. 
     
     
       25. The method of  claim 23 , wherein said removing step occurs prior to said cleaving at least a portion of the chemically fixed bonds. 
     
     
       26. A method of analyzing an analyte, the method comprising:
 providing a cellular sample comprising a chemically crosslinked analyte, wherein the sample is a formalin-fixed, paraffin-embedded tissue section; 
 removing the analyte from the paraffin of the formalin-fixed, paraffin embedded, tissue section; 
 reversing at least a portion of the chemical crosslinks in the crosslinked analyte and forming a decrosslinked analyte, wherein substantially no naturally occurring bonds in the analyte are broken during the reversal; and 
 analyzing the analyte by analyzing the decrosslinked analyte using mass spectrometry, wherein the analyte is a protein or a peptide. 
 
     
     
       27. The method of  claim 26 , wherein said removing step occurs prior to said reversing a least a portion of the chemical crosslinks.